Magnesium is an essential ion involved in many biochemical and physiological processes. Homeostasis of magnesium levels is tightly regulated and depends on the balance between intestinal absorption and renal excretion. However, little is known about specific proteins mediating transepithelial magnesium transport. Using a positional candidate gene approach, we identified mutations in TRPM6 (also known as CHAK2), encoding TRPM6, in autosomal-recessive hypomagnesemia with secondary hypocalcemia (HSH, OMIM 602014), previously mapped to chromosome 9q22 (ref. 3). The TRPM6 protein is a new member of the long transient receptor potential channel (TRPM) family and is highly similar to TRPM7 (also known as TRP-PLIK), a bifunctional protein that combines calcium- and magnesium-permeable cation channel properties with protein kinase activity. TRPM6 is expressed in intestinal epithelia and kidney tubules. These findings indicate that TRPM6 is crucial for magnesium homeostasis and implicate a TRPM family member in human disease.

Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg 2؉ -permeable cation channel TRPM7, resulting in the assembly of functional TRPM6͞TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6͞ TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.I nvestigations on Drosophila flies with impaired vision due to mutations in the transient receptor potential gene (trp) initiated a search for homologous proteins in mammals, leading to the discovery of three subfamilies of cation channels: TRPCs (canonical or classical TRPs), TRPVs (vanilloid receptor and related proteins), and TRPMs (melastatin and related proteins) (1, 2). TRPC channels mediate cation entry in response to phospholipase C activation, whereas TRPV proteins respond to physical and chemical stimuli, such as temperature, pH, and mechanical stress (3, 4). Within their respective subfamilies, TRPCs and TRPVs form homo-and heterotetramers displaying novel pore properties when compared to their homomultimeric counterparts (1, 5-9). The eight TRPM family members differ significantly from the aforementioned TRP channels in terms of domain structure, cation selectivity, and activation mechanisms (3, 10). Two TRPM proteins, TRPM6 and TRPM7, harbor serine͞threonine kinase domains in their C termini (11-16). Furthermore, TRPM7 displays unusual permeation properties by conducting a range of divalent metal ions including Mg 2ϩ and Mn 2ϩ (13,17,18).It was recently shown that autosomal recessive hypomagnesemia with secondary hypocalcemia (HSH) is caused by mutations in the TRPM6 gene (15,16). HSH is characterized by low serum Mg 2ϩ levels due to defective intestinal absorption or͞and renal wasting of Mg 2ϩ . Here we demonstrate that TRPM6 requires assembly with TRPM7 to form channel complexes in the cell membrane and that disruption of multimer formation by a mutated TRPM6 variant, TRPM6(S141L), results in human disease. MethodsMolecular Biology and Generation of TRPM6 Polyclonal Antisera. The cloning procedure of human TRPM6 isoforms (Table 1) as well as amplification of other TRPM cDNAs is described in detail in Supporting Methods, which is published as supporting information on the PNAS web site. For TRPM proteins C-terminally fused to cyan (CFP) or yellow (YFP) fluorescent proteins, STOP codons in TRPMs were replaced by XhoI restrictio...

Primary hypomagnesemia with secondary hypocalcemia is a rare autosomal recessive disorder characterized by profound hypomagnesemia associated with hypocalcemia. Pathophysiology is related to impaired intestinal absorption of magnesium accompanied by renal magnesium wasting as a result of a reabsorption defect in the distal convoluted tubule. Recently, mutations in the TRPM6 gene coding for TRPM6, a member of the transient receptor potential (TRP) family of cation channels, were identified as the underlying genetic defect. Here, the results of a TRPM6 mutational analysis of 21 families with 28 affected individuals are presented. In this large patient cohort, a retrospective clinical evaluation based on a standardized questionnaire was also performed. Genotype analysis revealed TRPM6 mutations in 37 of 42 expected mutant alleles. Sixteen new TRPM6 mutations were identified, including stop mutations, frame-shift mutations, splice-site mutations, and deletions of exons. Electrophysiologic analysis of mutated ion channels after heterologous expression in Xenopus oocytes proved complete loss of function of TRPM6. Clinical evaluation revealed a homogeneous clinical picture at manifestation with onset in early infancy with generalized cerebral convulsions. Initial laboratory evaluation yielded extremely low serum magnesium levels, low serum calcium levels, and inadequately low parathyroid hormone levels. Treatment usually consisted of acute intravenous magnesium supplementation leading to relief of clinical symptoms and normocalcemia, followed by lifelong oral magnesium supplementation. Serum magnesium levels remained in the subnormal range despite adequate therapy. This is best explained by a disturbed magnesium conservation in the distal convoluted tubule, which emerged in all patients upon magnesium supplementation. Delay of diagnosis resulted in permanent neurologic damage in three patients.

Background. Previous studies showed significant interaction between the local and systemic inflammatory response after severe trauma in small animal models. The purpose of this study was to establish a new combined trauma model in pigs to investigate fracture-associated local inflammation and gain information about the early inflammatory stages after polytrauma. Material and Methods. Combined trauma consisted of tibial fracture, lung contusion, liver laceration, and controlled hemorrhage. Animals were mechanically ventilated and under ICU-monitoring for 48 h. Blood and fracture hematoma samples were collected during the time course of the study. Local and systemic levels of serum cytokines and diverse alarmins were measured by ELISA kit. Results. A statistical significant difference in the systemic serum values of IL-6 and HMGB1 was observed when compared to the sham. Moreover, there was a statistical significant difference in the serum values of the fracture hematoma of IL-6, IL-8, IL-10, and HMGB1 when compared to the systemic inflammatory response. However a decrease of local proinflammatory concentrations was observed while anti-inflammatory mediators increased. Conclusion. Our data showed a time-dependent activation of the local and systemic inflammatory response. Indeed it is the first study focusing on the local and systemic inflammatory response to multiple-trauma in a large animal model.